...cleavage sequence, tRNAs, multiple individual promoters).  spCas9 and its gRNAs are also larger than their...and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed Lentiviral Expression..., and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection...expressed in 293 cells. This allows for a single promoter to drive expression of a crRNA array and doesn...arrays eliminates the need to include multiple promoters to drive crRNA expression and sequences that allow... Zetsche et al reverses the orientation of the promoter driving expression of the crRNA array (bolded ... Make sure to order 5’ phosphorylated oligos or treat with T4 PNK. Delivery options for Cpf1 multiplexing...

...at cancer cell metabolism. Cancer cells promote oxidative stress leading to the degradation of one key .... Unfortunately, as a whole, microenvironments promote tumor durability instead of repressing them, making...caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle progression and enhances tumor...signaling, possibly inducing the secretion of growth promoting proteins, such as human growth factor, vascular...Fibroblasts, or CAFs. CAFs are altered fibroblasts and promote the development and metastasis of tumors through...different organs. This means that exosomes not only promote microenvironment’s malignancy but also incite different...tumor’s evasiveness, evolution, and response to treatment. Some microenvironment cells improve tumor’s durability...

...most imporant is that, upstream of the Cbh promoter, this plasmid has a U6 promoter followed by BbsI restriction...SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified version...version of CAG promoter that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course...

...proteins (bEBPs) at a sequence upstream of the promoter called the upstream activating sequence (UAS). The...σ54-based transcription. Transcription from σ54 promoters requires binding of bacterial enhancer binding...gRNA complex which allows transcription from the promoter.  They found that the system is active in E. coli... and displays high dynamic range. However, the promoter in question must be σ54-dependent and the system...and further the study of natural σ54-dependent promoter systems. Find these CRISPR activation plasmids... reporter by inserting the NanoLuc luciferase downstream of the apoB gene. Using this reporter, the researchers...small-molecule probes to aid in the understanding and treatment of cardiovascular disease. Find the LipoGlo plasmids...

...plasmids used to express CRE under neuron-specific promoters (aCaMKII and hSyn). Created with BioRender.com...plasmids used to express Cre under neuron-specific promoters, including calmodulin-dependent kinase II alpha...excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized...expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability to induce CRISPRa... CRISPRa in primary rat neurons over other promoters, including ubiquitous ones such as EF-1α and CAG....consequently regulates neuronal physiology by promoting the development of neurons and synapses as well...cells to determine which transcription factors promote neuronal differentiation (Liu et al., 2018). The...

...shRNA under the control of a tet-responsive H1 promoter Trono 11651 pLVUT-tTR-KRAB 3rd Inducible expression...11795 pLL3.7 3rd Expresses shRNA under mouse U6 promoter; CMV-EGFP reporter cassette is included to monitor...silencing were added; Expresses shRNA under mouse U6 promoter; CMV-EGFP reporter cassette is included to monitor...with a chimeric 5’LTR, Any expression cassette (promoter and gene of interest) can be cloned into the plasmid...coexpression. Trono 17619 EF.CMV.RFP 2nd EF-1 alpha promoter for transgene and CMV drives expression of RFP...41393 pCW57.1 3rd Inducible lentiviral expression, TRE-gateway; PGK-rtTA-2A-puro. See article for more versions...

... cauliflower mosaic virus 35S promoter (CaMV 35S) or ubiquitin promoters (Belhaj et al., 2013). Addgene...achieved by placing the gRNA downstream of a plant-specific RNA pol III promoter, such as AtU6, TaU6, OsU6...consistent between plants and other organisms, but the promoter used to drive gRNA expression is dependent upon... gRNA” backbones which contain a plant pol III promoter and gRNA scaffold sequence and allow researchers...is commonly driven by plant-derived RNA pol II promoters which regulate expression of longer RNAs (such...expression). Examples of commonly used RNA pol II promoters for Cas9 expression include the ubiquitously expressing...Summary While different in many of the specifics - promoters used, precise protein sequences or domains, and...

...fairs are also an opportunity for the societies to promote their upcoming events and intro sessions, and give...Since 1996, Cambridge Biosoc’s goal has been to promote interest in the biological sciences amongst the...night. The society also visits high schools to promote chemistry degrees and provide advice to students... to support iGEM alumni-industry networking to promote the creation of start ups and biotech spin offs...Laureates, who speak about their work and current, trending topics in their area of research. The society ...

...tandem with a single promoter, but only the gene directly following the promoter is expressed. Cre-mediated... farther downstream in the construct. As RFP and OFP are directly downstream of the promoter in Brainbow...an RFP, CFP, and YFP downstream of a ubiquitous promoter. Zebrabow is an extremely versatile technology...to neurons by placing Brainbow downstream of a neural-specific promoter, or the entire fish could be labeled...Brainbow system are that it does not use ubiquitous promoters, meaning that not all cell types can be labeled...non-fluorescent YFP and activate fluorescence of the downstream OFP, GFP, or RFP. Cells that express the construct...adult fly. As a stop cassette is located directly upstream of the fluorescent protein genes, Cre recombination...

... the start of the gene to end up just downstream of the promoter found in the backbone. Ideally, the backbone... enzyme cut sites (restriction sites) downstream of the promoter as part of a multiple cloning site (MCS...place your gene in the proper orientation downstream of the promoter, you can add an EcoRI site just 5’ of... leaving the 5’ end of your gene just downstream of the promoter and placing the gene in the proper orientation...easily orient your gene insert with respect to the promoter. For example, let’s say your plasmid backbone ... on the left side of the image below. It has a promoter (blue arrow) followed by the restriction sites...of the backbone plasmid (see below). You should treat your digested backbone plasmid with a phosphatase...

...putting a gene of interest directly downstream of a tissue-specific promoter sounds simpler, this modular system...Gal4 expression can be regulated by any chosen promoter, and Bloomington’s Stock Center has fly lines ...lines expressing Gal4 under a variety of promoters resulting in tissue specific expression. To express YFG... to making a new fly with hedgehog under a new promoter each time a new hypothesis is tested. Controlling...gene expression. Thus, genes and siRNA encoded downstream of the UAS sequence are only expressed when Gal4...systems in Drosophila: A synthesis of time and space, Trends Genet. 20, 384–391 (2004). PubMed PMID: 15262411...I. Bartos, S. Marka, J. C. Kagan, Environmental Stress Causes Lethal Neuro-Trauma during Asymptomatic ...

...bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will... proteins from the same mRNA. We've discussed promoters before so in this blog post we’ll cover basics...screenable or selectable markers from a unique promoter, multicistronic plasmids ensure that any cells...originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic ... the upstream cistron will have a few additional residues added to the end, while the downstream cistron...scientists have reported lower expression of the downstream cistron due to factors such as the experimental...the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and...

...approximately 19 kb in length. It contains short promoter sequences at its 3’ and 5’ ends that are crucial..., the roles of specific viral sequences (e.g. promoters) can be studied by changing the minigenome cis-acting...minigenome) are under control of a T7 RNA polymerase promoter sequence. Thus, T7 RNA polymerase also needs to...leader region acts as a functional BRSV genome promoter." Journal of virology 73.1 (1999): 251-259. PubMed...Michael, et al. "The Ebola virus genomic replication promoter is bipartite and follows the rule of six." Journal...one developed for Zaire ebola virus (EBOV) are extremely useful, allowing researchers to study aspects ... systems, such as the one described here, are extremely useful research tools. In addition to the reduced...

...vitro models for lung disease. Darrell strives to promote open source biology in his own lab, at the Center...to help keep us focused on our core mission to promote scientific sharing. Listen to learn more about ... defines Open Science and how he practices and promotes open science in the lab - sharing reagents, cell...Darrell's ultimate goal - to develop therapies to treat disease in the lung. Chapter 2: Balancing research...Meidince - Understanding and developing means to treat disease through tissue repair, stem cells, gene ...

...TIR1 of the SCF-TIR1 ubiquitin ligase complex, promoting its interaction with the degron sequence and ultimately...studying the effects of endogenous looping on distal promoters and enhancers, or have attempted to engineer loops...LADL Anchors to genomic targets, such as a distal promoter and an enhancer. What makes LADL unique from previous...group targeted the enhancer region of Klf4 and the promoter region of Zfp462, which are over 500kB apart, ...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...

... gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene’s fly library web page... over 30 promoters and 140 genes, including constructs with unique tissue-specific promoters expressing...dCAS9-VPR expression is under the control of the TRE promoter, which allows for doxycycline-inducible expression...are cloned downstream of a minimal promoter sequence. Should one of these random stretches of DNA contain...its own transcription from the minimal promoter and its strength can be determined from its enrichment ...many different promoters, RBS’, coding sequences (CDS), and terminators of varying strengths. These can be...fluorophores, generate expression vectors with various promoters suitable for in vivo expression, and/or produce...

...cell-type specific promoters; however, those usually drive weak expression of downstream genes. Strong cell-specific...Optogenetic FLEx vectors contain a strong promoter upstream of an opsin gene (e.g., channelrhodopsin-2...cell-specific promoters, on the other side, are too long for rAAVs, which can only package sequences shorter... the antisense orientation with respect to the promoter to prevent its expression, and is flanked (“floxed...rAAVs expressing Cre under a specific cell-type promoter. Once injected into the brain, the viruses infect...thereby enabling its expression under a strong promoter only in specific neuronal cells. FLEx ensures,...for analyzing the neural circuits of behavior." Trends in cognitive sciences 15.12 (2011): 592-600. PubMed...

...technique is especially useful for introducing promoters, terminators, and other short sequences into the...creating a contiguous DNA fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein...

... Pro-Tips Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for 293T...fluorescent cells, these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...transcription start site, splice sites, exons, and/or promoters to knock out lncRNA function (Hazan and Bester...of introns or the removal of exons. Disrupting promoter or transcription start site function would also...effective in reducing off-target effects since promoter and enhancer sequences remain intact (Engreitz...Local regulation of gene expression by lncRNA promoters, transcription, and splicing. Nature. 2016;539...elements that are crucial for transcription and downstream function. Rather than attempting to cut out the...For example, inserting a polyadenylation signal downstream of the transcription start site will terminate... ncRNAs While CRISPR/Cas9 has proven to be an extremely useful system for modifying and assessing ncRNA...

... elements are cloned into a plasmid upstream of a minimal promoter linked to luciferase. Activation of...enzymatic activity is measured. The effect of promoters or enhancer regions on gene expression can be ...investigate the effect of regulatory elements, such as promoters, enhancers and untranslated regions, or the effect...Cofactors/Substrates Required Not Required Signal Strength Lower Higher Sensitivity Higher Lower Background...regulatory element (wild-type or mutant) is cloned upstream of the luciferase gene in the plasmid, which is...Expression of luciferase can be monitored as a downstream measurement of miRNA efficiency on a target sequence...The gene of interest is cloned into a plasmid downstream of luciferase containing a stop codon, which ...

...realized I wanted a career in science outreach: engaging the public to promote an awareness and understanding...to use to promote future attempts and learned a bit about recruiting scientists to outreach.  Try new ...spray paint to make a trophy for the winner. We promoted the contest for about two months, and we got one...network and graduate school resources daily to promote CINSO events and recruit speakers and participants... executive director of Central Indiana Science Outreach and a Postdoc at Indiana University School of ...founding a nonprofit called Central Indiana Science Outreach, or CINSO. We organize fun science events for ...supportive mentor and graduate office. I cannot stress how important they were to my success as a student...

...Effective science communication is required to promote public support for research and to keep useful ... a graduate student organization that works to promote science communication. Science in the News hosts...and SITN The Most Difficult thing Vini, Amy, and Outreach Scientist Jessica Welch Have Had to Explain Importance...

...plasmids, the MCS is often downstream from a promoter. Insert Gene, promoter or other DNA fragment cloned...cloned into the MCS for further study. Promoter Region Drives transcription of the target gene. Vital...

... a tool that can help you advance your career, promote career growth, and/or switch your field of work...is larger and there are more opportunities for promotion. It’s not uncommon for bachelor’s and master’s..., i.e. Principal Scientist or Scientist I/II. Promotions don’t happen overnight though, and it might take...but in time I realized its value. My master’s strengthened my critical thinking and problem solving abilities...

...design your donor template to include a T7 promoter upstream of the left homology arm, perform in vitro...transcription to create your ssDNA. Note that the T7 promoter will not be included in the final ssDNA template...GG); this design has been shown to increase T7 promoter activity. To avoid re-cutting by Cas9 after editing...containing the homology arms on both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best ...Addgene plasmids. Design PCR primers to add T7 promoter-left homology arm and right homology arm to the...template with 55-100 base homology arms upstream and downstream of the insertion site. If you are using...

... may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate... in this method include fluorescent proteins, promoters, enhancers, polyadenylation signals, inducible...Table 1). Each construct was flanked with a CMV promoter (to drive high-level expression) and a polyA signal...multiple identical polyadenylation signals or promoters Gibson et al., 2009 MXS-Chaining, BioBricks,...and different methods are suited for particular downstream applications. The tradeoffs between various cloning...

...induce transcription at CpG regions of various promoters. Other researchers additionally fused a TAL effector...: dCas9-p300 adds H3K27ac marks to histones in promoter and enhancer regions. These marks are associated...Figure 2: dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation is ...Figure 3: dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation is associated...-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517...in the DNA oxidation and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal...

...optimism they encapsulate, and the solutions they promote help motivate you to spend a little time advocating...fight is far from over here at home.  The work of promoting and defending the role of science in our democracies... take in abusing climate science skepticism to promote harmful policies that benefit polluting industries.In...marching in and of itself will be enough. IT WON’T! Promoting the incorporation of science in policy, demanding... that uses yeast cells to study how DNA damage promotes cancer. She is also a science communicator, UBC...Hundreds of scientists and supporters in Montreal took to the streets on April 22 to join in the March for...at the Goodman Cancer Research Centre at McGill University in Montreal, Canada.  Outside the lab, she ...

...Regulated Cre expression – placing Cre downstream of promoters that are active only in certain cell or...examined a number of important plasmid elements – promoters, origins of replication, protein tags, and antibiotic...Cre under a variety of ubiquitous and regulated promoters, and many loxP-containing alleles have also been...often called a “lox-stop-lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression...